Process for preparation of high molecular weight cell-associated protein of Campylobacter pylori and use for serological detection of Campylobacter pylori infection

ABSTRACT

An antigen for the detection of Campylobacter pylori infections and an assay for the serological detection of Campylobacter pylori. The antigen includes high molecular weight cell-associated proteins purified from Campylobacter pylori. The antigen can be used in a variety of assays including radioimmunoassay, ELISA, latex agglutination, complement fixation, and indirect hemagglutination. Furthermore, the antigens can be combined with a solid support in kit form.

.Iadd.The work herein was supported by grants from the United StatesGovernment. .Iaddend.

FIELD OF THE INVENTION

This invention relates to high molecular weight cell-associated proteinsused as antigen for the detection of antibody to Campylobacter pyloriinfection. It is useful both for the preparation of the antigen as wellas in the detection and monitoring of the infection.

BACKGROUND OF THE INVENTION

Campylobacter pylori (C. pylori) was first isolated in 1982. It is nowknown to be an important cause of gastritis and has been associated withduodenal ulcer, gastric ulcer, dyspepsia and gastric carcinoma. Sincethe discovery in 1982 there has been a tremendous worldwide interest inC. pylori and in trying to delineate its actual role in gastric diseaseand the formation of ulcers. Despite the numerous studies showing aclose association between C. pylori and abnormal gastric pathology,there is insufficient evidence to determine conclusively whether theorganism is pathogenic or opportunistic. Nevertheless, the presence ofC. pylori is an important consideration in treating gastric disease.

Patients colonized with C. pylori elicit a specific antibody responsewhich is potentially useful as a diagnostic aid and for monitoring thedisease state during treatment. Consequently, many systems have beendeveloped to detect serum anti C. pylori antibodies. However,preliminary studies suggest that C. pylori displays antigeniccross-reactivity with the thermophilic campylobacteria C. jejuni and C.coli. This cross-activity results in lack of specificity.

In attempts to avoid the problems associated with cross-reactivity,investigators have extensively studied the acid extractable surfaceproteins and outer membrane proteins of C. pylori. Newall, D. G.,Journal of General Microbiology 133:163-170 (1987); and Perez-Perez, G.I. and Blaser, M. J., Infection and Immunity 55:1256-1263 (1987). Newalldemonstrated that there existed acid extractable proteins in themolecular range of 20,000 to .Iadd.100,000 daltons which were unique toC. pylori. However, .Iaddend.some of these proteins were similar toproteins of C. jejuni and many also showed cross-reactivity with C.jejuni. At least one major antigen (approximately 60,000 daltons) showedonly minimum cross-reactivity with C. jejuni, however, there still wassome cross-reactivity. On the other hand, Perez-Perez showed that anantigen of about 62,000 daltons had significant cross-reactivity. C.pylori is capable of elicting both a systemic and local antibodyresponse in patients with chronic gastritis, however, thissecretory-antibody response does not appear to eliminate thecolonization. Rathbone, B. J. et al., Gut, 27:642-647 (1986). Rathboneet al used the whole organism in the immunological assay.

Other studies using the immunoblot technique show that C. pylori has anumber of immuno-reactive components in the range of 100,000 daltons orless. Kaldor, J. et al. The Medical Journal of Australia 145:133-135(1986).

Whole organism ELISA assays detect C. pylori antibody but still areunable to solve the cross-reactivity problem. Morris et al. The NewZealand Medical Journal 99:657-659 (1986).

Acid-glycine extracts of C. pylori detect antibodies using ELISAtechniques. However, a number of false positives and false negativesexist. Although the relative number of each false result can beregulated by adjusting the cut off point, there is still significantoverlap between the groups. Goodwin et al. The Journal of InfectiousDisease 155:488-494 (1987). Similar results are found using complementfixation, bacterial aglutination and immunoblotting. Jones et al.General Clinical Pathology 37:1002-1006 (1984) and Jones et al. J. ofEd. Microbiology 22:57-62 (1986). Acid washed fractions show similarresults in both complement fixation and SDS-PAGE immunoblots. Wulffen etal. Journal of Clinical Microbiology 24:716-720 (1986).

Numerous reports exist showing C. pylori antibodies in the serum ofaffected humans. All of the studies have dealt with the outside surfaceof the microorganism. In these test systems, the antigen is either thewhole organism or sub parts of the flaggella and outside membrane in themolecular weight range of about 100,000 daltons or less. None of thesestudies are adequate to allow accurate detection of the infection. Thereis significant misclassification, both false positive and falsenegative, as well as significant cross-reactivity with other organisms,such as C. jejuni and C. coli. Thus, there exists a need for a quickimmunological method to specifically detect the C. pylori antibody. Thepresent invention meets this need. The present invention describes a newand accurate serological assay for the diagnosis of C. pylori infection.Previously published results used lower molecular weight compounds andhad significant levels of cross-reactivity with other bacteria. No otherassay has the same overall reliability (sensitivity plus specificity).

The symptom dyspepsia is associated with large health care expendituresthroughout the western world. Although accurate statistics as to thefrequency of dyspepsia are difficult to obtain, recent studies haveshown it to be a common problem. In England, for example, it has beenestimated that approximately 1% of patients served by generalpractitioners will present each year with the primary complaint ofdyspepsia. The costs of dyspepsia are many and include: (i) those fordrugs such as antacids or H₂ -receptor antagonists (sales of cimetidineand ranitidine were more than 2 billion dollars); (ii) charges fordiagnostic evaluations such as barium upper gastrointestinal series orfiberoptic endoscopy and (iii) cost associated with time off from work.The effects of dyspepsia on drug use were studied by evaluating patientsin Sweden in whom a clinical diagnosis of gastritis or non-ulcerdyspepsia was made. Tyllstrom et al. Scand. J Gastroenterol 1984,19:755-60.

Tyllstrom found that antacid or H₂ -receptor antagonist therapy wascommon among these patients. In fact, most patients who visited aphysician were given a prescription. This result is similar to data fromBritain in which 91% of such patients reported regular use of antacids.Tyllstrom calculated that 1% of the entire population of Sweden wastaking a daily dose and that non-ulcer dyspepsia was a primaryindication for cimetidine use, accounting for 35% of the prescriptions.There was also noted an increasing trend in the percentage of patientstreated with cimetidine.

Because of the high incidence and cost of gastric problems and ulcers inwestern society, the ability to detect and monitor the treatment ofthese diseases is highly desirable. Thus the present invention isimportant in its ability to specifically detect C. pylori which isassociated with these diseases and whose disappearance is associatedwith clinical improvement.

SUMMARY OF THE INVENTION

An object of the present invention is the isolation and purification ofantigens from the high molecular weight cell-associated proteins of C.pylori.

An additional object of the present invention is a method for detectingC. pylori infection in humans.

A further object of the present invention is a diagnostic kit.

Thus, in accomplishing the foregoing objects, there is provided inaccordance with one aspect of the present invention antigens from thehigh molecular weight cell-associated proteins (HM-CAP) of C. pylori,the antigens in substantially purified form having a molecular weight ofabout 300,000 to 700,000 daltons, a PI on isoelectric focusing of about5.9 to 6.3, being soluble in phosphate-buffered saline and tris-chloridebuffers. In one preferred embodiment the antigen demonstrates ureaseactivity.

Another embodiment includes a serological assay for the detection of C.pylori infection in humans comprising combining the antigens isolatedfrom HM-CAP with a serum sample to be tested according to theimmulogical method selected from the group consisting of enzyme-linkedimmunosorbent assay, radioimmuno assay, complement fixation, latexagglutination, and passive hemagglutination test using HM-CAP coatederythrocytes pre-treated (activated) by glutaraldehyde or tannic acid.

In one embodiment an enzyme-linked immunosorbent assay is used. Thisassay includes immobilizing the antigen on a solid phase support, addingserum sample to the immobilized antigen, incubating the serum sample andthe immobilized antigen to form an antigen-antibody complex. Adding anenzyme-conjugated anti-human IgG to the antigen-antibody complex andincubating to form an antigen-antibody enzyme-conjugated anti-human IgGcomplex. In a preferred embodiment the enzyme can include alkalinephosphatase, horseradish peroxidase or beta-galactosidase. When alkalinephosphatase is used, para-nitrophenyl phosphate (enzyme substrate) isadded to the complex. This substrate reacts with the alkalinephasphatase yielding a color which can be measured to determine theamount of antibody.

Another embodiment includes a kit which is comprised of the antigens ofhigh molecular weight cell-associated proteins of C. pylori immobilizedon a solid phase support.

Other and further objects, features and advantage will be apparent fromthe following description of the presently preferred embodiments of theinvention given for the purpose of disclosure when taken in conjunctionwith the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be more readily understood from a reading of thefollowing specification and by reference to the accompanying drawings,forming a part thereof, where examples of embodiments of the inventionare shown and wherein:

FIG. 1 is a typical elution profile of crude HM-CAP applied to anagarose A-5m column.

FIG. 2 is a typical elution profile of HM-CAP preparation from Broth-.Iadd.o---o- .Iaddend.and plate -.Iadd.o---o- .Iaddend.grown bacteriashowing the region of urease activity.

FIG. 3 compares four different positive and negative sera using an ELISAassay with antigen from plate grown bacteria to detect anti-C. pylori.

FIG. 4 compares four different positive and negative sera using an ELISAassay with antigen from both grown bacteria to detect C. pylori.

DETAILED DESCRIPTION

The drawings are not necessarily to scale and certain features of theinvention may be exaggerated in scale or shown in schematic form in theinterest of clarity and conciseness. It will be readily apparent to oneskilled in the art that various substitutions and modifications may bemade to the invention disclosed herein without departing from the scopeand spirit of the invention.

Antigens from the high molecular weight cell-associated proteins(HM-CAP) of C. pylori are in substantially purified form having amolecular weight of about 300,000 to 700,000 daltons, a PI onisoelectric focusing of about 5.9 to 6.3, being soluble in commonly usedbuffered solutions including PBS (phosphate-buffered saline includingabout 0.05M phosphate buffer, about 0.85% NaCl at about pH 7.2) orTris-chloride buffer (about 0.05M Tris, pH about 8.0). The proteincomponents are detectable by absorption at 280 nm, lowering proteinassay and by staining gels with Coomasee Blue. HM-CAP is extracted(solubilized) by treatment of C. pylori cells with n-octyl-glucoside(NOG). NOG extracts membrane and surface proteins without breaking thecells. In the preferred embodiment these antigens demonstrate ureaseactivity.

When C. pylori cells are harvested and washed, urease activity remainsbound to the bacterial cells. After sonication and centrifugation ureaseenzyme activity resides in the pellet providing further evidence thatthe protein with urease activity is associated with the outer surface ofthe membrane. The "proteins associated with the outer surface of themembrane" refers to proteins which are either in the membrane or are onthe surface of the membrane. Furthermore, disruption of the cell surfacewithout breaking the cell releases urease activity into the supernatantfraction.

The antigens can be prepared by a variety of methods. In the preferredembodiment, C. pylori is first grown on blood agar plates. The bloodagar plates are prepared with about 7% fresh (not more than eight daysold) horse blood and DIFCO brain-heart fusion base. After incubating thecultures for about 48 hours at about 37° C. in about 12% CO₂ and about100% humid atmosphere, the C. pylori are harvested from the plates. Theharvested bacteria are washed with PBS centrifuged at about 8,000 rpmfor about 12 minutes. This is repeated at least twice.

In another embodiment the C. pylori are grown in a broth medium composedof DIFCO brain-heart infusion broth containing about 10% horse serum,about 0.03% purified rabbit hemoglobin and about 0.15% DIFCO yeastextract. For broth cultures, the inoculum is prepared from a blood agarplate (as described above). Incubation conditions are the same as forblood agar plate cultures. Bacteria from broth are harvested bycentrifugation at about 8,000 rpm for about 12 minutes and then thewashing procedure as described for plate-grown bacteria is followed.

Next, the washed C. pylori cells, whether from broth or agar plates, areextracted by resuspending the washed bacteria in about 1% solution ofn-octyl-glucoside in PBS, about pH 7.2, using about 2.5 ml per 1.0 ml ofpacked cells. After extraction for 20 minutes at room temperature theextraction suspension is centrifuged at about 15,000 rpm for 15 minutes.The supernatant is removed and dialyzed for 18 to 24 hours against 1,600volumes (about 4 liters) against one-half concentration PBS containing0.024% sodium azide as a preservative. The dialysate is centrifuged atabout 18,000 rpm for about 15 minutes. The pellet material is discardedand the supernatant is saved. The supernatant contains the crude(HM-CAP). The crude HM-CAP preparation is placed on a Agarose A-5 mcolumn and eluted with about 0.05M Tris-Cl buffer, at about pH 8.0containing about 0.025% sodium azide. The column is about a 1.6 by 100centimeter column. Approximately 2.5 milliliter fractions from thecolumn are collected and monitored. The optical density of thesefractions is determined at 280 nm and urease activity determined byassay with urea as substrate. The fractions (about 6 to 8) which containmaximum urease activity are pooled. These fractions which are pooledrepresent the molecular weight range of about 300,000 to 700,000daltons. The HM-CAP preparation at this point contains at least twoseparate proteins.

FIG. 1 shows the results obtained when 2.5 ml of the crude HM-CAPpreparation is passed through an agarose A-5 m column. The peak of 280nm absorbing material at fractions 47-49 coincides with the peak ofurease activity and is closely followed by another peak of 280 nmabsorbing material of lower molecular weight at fractions 51-52. Thesetwo peaks have considerable overlap and the partially purified HM-CAP,fractions 47-49, contains at least two and probably more molecularspecies. Further separations can be undertaken to separate theseindividual proteins.

Molecular weight (MW) was determined by eluting proteins of knownmolecular weights through the same agarose A-5m column and noting theirelution positions, for example, thyroglobulin (MW 669,000) at fraction46; aproferritin (MW 443,000) at fraction 51; yeast alcoholdehydrogenase (MW 150,000) at fraction 55; and bovine serum albumin (MW66,000) at fraction 62. From these protein standards it is can becalculated that partially purified HM-CAP contains molecular species inthe range of 300,000 (fraction 53) to 700,000 (fraction 46) molecularweight.

The antigenic activity of the individual protein fractions is shown inFIGS. 2-4. HM-CAP fractions were selected and pooled on the basis ofurease activity and used as antigen in an ELISA assay. These antigenswere extremely effective in detecting the presence, or absence, ofanti-C. pylori serum IgG antibody. The presence or absence of theorganism correlated with the presence or absence of the antigen. Thiscorrelation was confirmed by detecting the C. pylori organism with otherless convenient but recognized methods. Two batches of C. pylori weregrown, one using the plate method and one using the broth method, todetermine (1) how much of this specificity could be accounted for byantigens other than urease and (2) whether the method of growth of thebacteria (plates versus liquid media, or broth) for antigen productionhas any effect on the specificity of the ELISA. Crude HM-CAP wasprepared from both batches of bacteria and individually passed throughthe same agarose A-5m column. The elution diagrams are shown in FIG. 2.

In both cases two or more column fractions were pooled together tocreate the eight different pools designated in FIG. 2. Proteindeterminations were performed on each pool so that microtiter platescould be coated with an equivalent amount of protein (100 microliters ofantigen at 0.007 mg protein per ml) from each of the pools. The proteindetermination can be seen in the following table.

    ______________________________________                                        Protein In Agarose Fraction Pools Before Dilution                             Pool      Fractions      Mg/Ml Protein                                        Number    Pooled         Broth   Plate                                        ______________________________________                                        1         53-58          0.28    0.16                                         2         55-56          0.38    0.19                                         3         57-58          0.26    0.16                                         4         59-60          0.22    0.14                                         5         61-62          0.24    0.20                                         6         63-64          0.20    0.15                                         7         59-64          0.28    0.24                                         8         53-64          0.28    0.20                                         ______________________________________                                    

Pool 2 is equivalent to the HM-CAP antigen preparation used in thestandardized assay. Four ELISA-positive and four ELISA-negative serumsamples were selected at random and used to perform the ELISA assays.The results are shown in FIGS. 3 and 4.

Examination of FIGS. 3 and 4 demonstrate that Pools 1-4, representingagarose fractions 53-60, provide the greatest differentiation betweenELISA-positive and ELISA-negative sera, as compared to pools 5-8. Pools5-8 show some selectivity as test antigens and thus suggest that it isnot necessary to further purify HM-CAP in order to have a C.pylori-specific ELISA assay.

These results indicate that the selection of the urease-positive columnfractions for use as HM-CAP antigens in the C. pylori-specific ELISAassay provides adequate sensitivity and selectivity. Therefore, furtherseparation into specific components is not necessary. Additionally,plate-grown and broth-grown bacteria are equally useful as the source ofHM-CAP for ELISA antigens. The larger yield of urease protein in thebroth-grown batch of HM-CAP (see FIG. 2) most likely accounts for thefact that pools 5-8 of the broth-grown antigen performed better thanpools 5-8 of the plate-grown antigen due to less efficient separation ofthe two major peaks.

Thus FIGS. 3 & 4 show that the mixture of at least two proteins is justas effective in detecting C. pylori antibodies in serum as proteinindividually. Although these data show that the higher molecular weightcomponent, coinciding with urease activity, is a better antigen than thelower weight antigen they also suggest that the mixture is as good ifnot better in determining C. pylori infection. Thus, although theproteins can be further purified into individual components, stoppingthe purification prior to the separation of the mixture is sufficient.

A variety of methods can be used to detect C. pylori antibody in theserum. One skilled in the art will readily recognize that enzyme-linkedimmunosorbent assays (ELISA), radioimmuno assays (RIA), complementfixation, latex bead agglutination, immunoblot assays, and passiveinmugglutination can all be used. In a preferred embodiment, theserological assay involves the ELISA method. This assay includesimmobilizing the HM-CAP antigen on a solid phase support. After theantigen has been immobilized a serum sample to be tested is combinedwith the immobilized antigen and they are incubated for about 90 minutesat room temperature and under humid conditions. An antigen-antibodycomplex forms during the incubation. After the antigen antibody-complexis formed, alkaline-phosphatase-conjugated anti-human IgG is added tothe antigen-antibody complex on the solid phase support and incubatedfor approximately 90 minutes, at room temperature under humid conditionsto form an antigen-antibody-alkaline-phosphatase-conjugated anti-humanIgG complex. A variety of substrates can be used to determine the amountof binding which has taken place. In a preferred embodiment,para-nitrophenyl phosphate is added to this complex and the resultingyellow product is measured to determine the amount of antigen-antibodycomplex formed, and thus the amount of antibody present in the serum.The above mentioned assay methods can also be used to monitor thetreatment of C. pylori infection by collecting serial serum samples fromtreated subjects and performing said methods on each sample.

EXAMPLE 1

ELISA technique: Coat microtiter plate wells with 100 microliters ofHM-CAP antigen diluted with PBS to approximately 0.007 milligrams permilliliter protein. In one embodiment a standard 96 well plate is used.After about 18 to 24 hours at 37° C. in a humid chamber, the antigenadheres to the plastic surface, forming a non-specific, permanentbinding. The excess plastic protein-binding sites are blocked byincubating about 1% BSA (Bovine serum albumin) in PBS for about 30minutes at approximately 37° C. in a humid chamber. The excess BSA isremoved by washing three times with PBST (PBS Containing about 0.02%Tween-20). Next, about 100 microliters per well of serum is added indilutions of either 1:50 or 1:100 in PBS. After incubation at roomtemperature, in a humid chamber for about 90 minutes, the excessantibodies are removed by washing three times with PBST. About 100microliters per well of a calibration dilution of conjugate (goatanti-human IgG antibodies conjugated to the enzyme alkaline phosphatase)are added, and the mixture is incubated at room temperature in a humidchamber for about 90 minutes. After removing excess conjugate, i.e.,unreacted conjugate, by washing three times with PBST, about 100microliters per well of alkaline phosphatase substrate, for examplepara-nitrophenyl phosphate, are added. After additional incubation forabout 60 minutes, in a humid chamber the yellow colored enzyme productis measured. For each microtiter plate includes a number of controls,for example, known ELISA-positive serum, known ELISA-negative serum andreaction blank. An optical density value of about 0.200 or higher is apositive result. The following results were observed:

    ______________________________________                                        DIAGNOSIS OF C. PYLORI INFECTION                                                                  +    -                                                    ______________________________________                                        .Iadd.HM-CAP ELISA.Iaddend.                                                                 +           113     3                                           .[.HM-CAP ELSIA.].                                                                          -            3     90                                           ______________________________________                                    

C. pylori infection was diagnosed as either positive or negative byhistological examination and culture of biopsy material, or by the ¹³ Curea breath test.

The HM-CAP ELISA assay detected 113 of the 116 samples from individualswith C. pylori infection. This is a specificity of 97.4%. Thesensitivity of the assay is determined by looking at the negative HM-CAPELISA result. The results show 90 of 93 C. pylori negative individualswere detected. This is a sensitivity of 96.8%. The overall reliabilityof the test is 97.1% (203 out of 209 samples were accurately predicted).These data strongly indicate that there are few, if any,misclassifications using the ELISA assay.

EXAMPLE 2

KIT: A Kit is prepared by incubating the HM-CAP antigen on a solid phasesupport. The solid phase support can be any charged membrane or plasticmaterial. The solid phase support-antigen complex can then be packagedindividually or in multiple combinations. The kit can also includecontrols for false positives and false negatives, and reagents. The kitcan be used to detect 1 sample or multiple samples.

EXAMPLE 3

Latex agglutination assay: Antibodies against C. pylori can be measuredin serum sample with HM-CAP coated latex bead particles. Additionally,the presence of antigens can be measured by coating the latex particleswith monospecific antibody (anti-HM-CAP). Particles of polyvinyl ortoluene latex of about 0.77 micron diameter or polystyrene latexparticles (beads) measuring about 0.81 to 1.77 microns can be coatedwith HM-CAP. About 2.0 ml of the latex particles are suspended inapproximately 20 ml of distilled water, mixed and filtered through aWhatman No. 40 filter paper. After adjusting the filtrate to about 2.0optical density at a wavelength of 640 nm in phosphate-buffered saline,pH about 7.2., or equivalent buffer, about 0.1 ml of the latexsuspension is diluted with about 5.0 ml of PBS. About 0.5 of a 0.5%antigen solution is added to the diluted latex suspension. This mixtureis then incubated at about 37° C. for 30 minutes. The latex particlesare then washed twice, each time with ten volumes of PBS. The finalsuspension is adjusted to an optical density of 0.3 using 0.1M glycinebuffer containing 0.1% bovine serum albumin. In the assay equal volumesof coated latex particles and serum dilutions in 0.1M glycine buffer aremixed. Control tubes receive saline instead of serum. Tubes areincubated at 50° C. for 2 hours, centrifuged at 15,000×g for 3 minutesand gently tapped. The degree of clumping is noted. Clumping is due tothe aggregation (agglutination) of the beads via the antigen-antibodycomplex formation.

EXAMPLE 4

Radiommunoassay: A 96-well microtiter plate is coated with about 100microliters of an optimum concentration of antigen. After incubation atabout 37° C. for about 18-24 hours, the excess binding sites in thewells are blocked with 1% BSA in PBS, or equivalent. About 100microliters per well of appropriate dilution(s) of serum to be testedare added to duplicate wells and incubated at room temperature for about2 hours. After washing the plates numerous times with PBST, 100microliters per well of optimal dilution of goat or rabbit anti-humanIgG, which has been labeled with 125-iodine, is added. The microliterplates are incubated for about 4 hours at room temperature, washednumerous times with PBST and air dried. The wells are counted in a gammacounter to determine the amount of radioactivity remaining in each wellat the end of the test. Positive and negative sera are included on eachplate as controls. The procedure is standardized to determine whichvalues differentiate between a positive and a negative serum.

One skilled in the art will .[.really.]. .Iadd.readily.Iaddend.appreciate the present invention is well adapted to carry outthe objects and obtain the ends and advantages mentioned, as well as,those inherent therein. The methods, procedures and techniques describedherein are presently representative of the preferred embodiments, areintended to be exemplary, and are not intended as limitations on thescope. Changes therein and other uses will occur to those skilled in theart which are encompassed within the spirit of the invention or definedby the scope of the appended claims.

What is claimed is:
 1. A composition comprising substantially purifiedantigens from the high molecular weight cell-associated proteins ofCampylobacter pylori, said antigens:having a molecular weight of about300,000 to 700,000 daltons as determined by agarose A-5 m column; havinga PI on isolectric focusing of about 5.9 to 6.3; having ureaseactivity.Iadd.; .Iaddend. being soluble in PBS and Tris-chloridebuffers; being derived from the outer surface of the membrane ofCampylobacter pylori; and being solubilized from the outer surface ofthe membrane with n-octyl-glucoside.
 2. A serological assay for thedetection of Campylobacter pylori infection, comprising, combining theantigens of claim 1 with a serum sample to be tested according to amethod selected from the group consisting of enzyme-linked immunosorbentassay, radioimmunoassay, complement fixation, indirect hemagglutinationand latex agglutination wherein measurement of an antigen-antibodycomplex is proportional to the amount of Campylobacter pylori in theserum sample.
 3. The serological assay of claim 2, wherein said methodis the enzyme-linked immunosorbent assay, which includes the stepsof:adding serum sample to an antigen immobilized on a solid phasesupport; incubating the mixture of serum sample and immobilized supportto form an antigen-antibody complex; adding enzyme-conjugated anti-humanIgG to said antigen-antibody complex; incubating the antigen-antibodycomplex and enzyme-conjugated anti-human IgG mixture to form anantigen-antibody-enzyme-conjugated anti-human IgG complex; addingsubstrate to the antigen-antibody-enzyme-conjugated anti-human IgGcomplex; and measuring the product or the change in the substrate todetermine the amount of said antibody; wherein the product or change inthe substrate measured is proportional to the amount of Campylobacterpylori in the serum sample.
 4. The serological assay of claim 3, whereinthe enzyme is selected from the group consisting of alkalinephosphatase, horseradish peroxidase and beta galactosidase.
 5. A methodof monitoring the treatment of Campylobacter pylori infection,comprising, collecting serial serum samples from the treated subject andrepeating the steps of claim 3 on each sample.
 6. The serological assayof claim 2, wherein said method is the radioimmunoassay, which includesthe steps of:adding serum sample to a well coated with antigen;incubating said serum sample in said coated well to form anantigen-antibody complex; adding radioactive labeled anti-human IgG;incubating the mixture of the antigen-antibody complex and anti-humanIgG to form an antigen-antibody-anti-human IgG complex; and measuringthe amount of radioactivity bound in the antigen-antibody-anti-human IgGcomplex; wherein the amount of radioactivity bound is proportional tothe amount of Campylobacter pylori in the serum sample.
 7. Theserological assay of claim 2, wherein said method is latexagglutination, which includes the steps of:adding serum sample to latexbeads coated with antigen; incubating the serum sample and coated latexbeads; and measuring the degree of clumping; wherein the degree ofclumping is proportional to the amount of Campylobacter pylori in theserum sample.
 8. A kit for determining the presence of Campylobacterpylori antibody, comprising, a container having the antigens of claim 1immobilized on a solid phase support.
 9. The kit of claim 8, furthercomprising, .Iadd.a container having .Iaddend.false negative controlsand .[.a control having.]. false positive controls. .Iadd.
 10. Acomposition comprising substantially purified antigens from the highmolecular weight cell-associated proteins of Campylobacter pylori, saidantigens:having a molecular weight of about 300,000 to 700,000 daltonsas determined by agarose A-5 m column; having a PI on isolectricfocusing of about 5.9 to 6.3; having urease activity; being soluble inPBS and tris-chloride buffers; being derived from the outer surface ofthe membrane of Campylobacter pylori; and being capable of beingsolubilized from the outer surface of the membrane with n-octyl-glucose..Iaddend. .Iadd.
 11. A serological assay for the detection ofCampylobacter pylori infection, comprising, combining the antigens ofclaim 10 with a serum sample to be tested according to a method selectedfrom the group consisting of enzyme-linked immunosorbent assay,radioimmunoassay, complement fixation, indirect hemagglutination andlatex agglutination wherein measurement of an antigen-antibody complexis proportional to the amount of Campylobacter pylori in the serumsample. .Iaddend. .Iadd.12. The serological assay of claim 11, whereinsaid method is the enzyme-linked immunosorbent assay, which includes thesteps of:adding serum sample to an antigen immobilized on a solid phasesupport; incubating the mixture of serum sample and immobilized supportto form an antigen-antibody complex; adding enzyme-conjugated anti-humanIgG to said antigen-antibody complex; inhibiting the antigen-antibodycomplex and enzyme-conjugated anti-human IgG mixture to form anantigen-antibody-enzyme-conjugated anti-human IgG complex; addingsubstrate to the antigen-antibody-enzyme-conjugated anti-human IgGcomplex; and measuring the product or the change in the substrate todetermine the amount of said antibody; wherein the product or change inthe substrate measured is proportional to the amount of Campylobacterpylori in the serum sample. .Iaddend. .Iadd.13. The serological assay ofclaim 12, wherein the enzyme is selected from the group consisting ofalkaline phosphatase, horseradish peroxidase and beta galactosidase..Iaddend. .Iadd.14. A method of monitoring the treatment ofCampylobacter pylori infection, comprising, collecting serial serumsamples from the treated subject and repeating the steps of claim 12 oneach sample. .Iaddend. .Iadd.15. The serological assay of claim 11,wherein said method is the radioimmunoassay, which includes the stepsof: adding serum sample to a well coated with antigen; incubating saidserum sample in said coated well to form an antigen-antibody complex;adding radioactive labeled anti-human IgG; incubating the mixture of theantigen-antibody complex and anti-human IgG to form anantigen-antibody-anti-human IgG complex; and measuring the amount ofradioactivity bound in the antigen-antibody-anti-human IgG complex;wherein the amount of radioactivity bound is proportional to the amountof Campylobacter pylori in the serum sample. .Iaddend. .Iadd.16. Theserological assay of claim 11, wherein said method is latexagglutination, which includes the steps of: adding serum sample to latexbeads coated with antigen; incubating the serum sample and coated latexbeads; and measuring the degree of clumping; wherein the degree ofclumping is proportional to the amount of Campylobacter pylori in theserum sample. .Iaddend. .Iadd.17. A kit for determining the presence ofCampylobacter pylori antibody, comprising, a container having theantigens of claim 10 immobilized on a solid phase support. .Iaddend..Iadd.18. The kit of claim 17, further comprising, a container havingfalse negative controls and false positive controls. .Iaddend.